The present invention relates to an apparatus for automatically connecting an electrode to a cell. Particularly, the present invention provides an apparatus for carrying out patch clamp techniques utilized to study electrical activities, such as ion transfer channels, in biological membranes. The present invention broadly refers to a novel electrophysiology drug handling and application set up for screening chemical substances or compounds, such as high throughput, requires a low volume of solution and sample to be tested. The invention further relates to methods for utilizing the apparatus of the invention, such as in large scale screening programs in the pharmaceutical industry.
The general idea of electrically isolating a patch of membrane using a micropipette and studying the ion channels in that patch under voltage-clamp conditions was outlined by Neher, Sakmann, and Steinback in xe2x80x9cThe Extracellular Patch Clamp, A Method For Resolving Currents Through Individual Open Channels In Biological Membranesxe2x80x9d, Pflueger Arch. 375; 219-278, 1978. They found that, by pressing a pipette containing acetylcholine (ACh) against the surface of a muscle cell membrane, they could see discrete jumps in electrical current that were attributable to the opening and closing of ACh-activated ion channels. However, they were limited in their work by the fact that the resistance of the seal between the glass of the pipette and the membrane (10-50 Mxcexa9) was very small relative to the resistance of the channel (xe2x88x9210 Gxcexa9). The electrical noise resulting from such a seal is inversely related to the resistance and was large enough to obscure the currents through ion channels, the conductances of, which are smaller than that of the ACh channel. It also prohibited the clamping of the voltage in the pipette to values different from that of the bath due to the large currents through the seal that would result.
It was then discovered that by fire polishing the glass pipettes and applying gentle suction to the interior of the pipette when it made contact with the surface of the cell, seals of very high resistance (1-100 Gxcexa9) could be obtained, which reduced the noise by an order of magnitude to levels at which most channels of biological interest can be studied and greatly extended the voltage range over which these studies could be made. This improved seal has been termed a xe2x80x9cgiga-sealxe2x80x9d, and the pipette has been termed a xe2x80x9cpatch pipettexe2x80x9d. A more detailed description of the giga-seal may be found in: O. P. Hamill, A. Marty, E. Neher, B. Sakmann and F. J. Sigworth: Improved patch-clamp techniques for high resolution current recordings from cells and cell-free membrane patches. Pflxc3xcgers Arch. 391, 85-100, 1981. For their work in developing the patch clamp technique, Neher and Sakmann were awarded the 1991 Nobel Prize in Physiology and Medicine.
Ion channels are transmembrane proteins, which catalyze transport of inorganic ions across cell membranes. The ion channels participate in processes as diverse as generating and timing of action potentials, snaptic transmission, secretion of hormones, contraction of muscles, etc. Many drugs exert their specific effects via modulation of ion channels. Examples are antiepileptic compounds like phenytoin and lamotrigine, which block voltage dependent Na+-channels in the brain, antihypertensive drugs like nifedipine and diltiazem, which block voltage dependent Ca2+-channels in smooth muscle cells, and stimulators of insulin release like glibenclamide and tolbutamnide, which block an ATP-regulated K+-channel in the pancreas. In addition to chemically induced modulation of ion-channel activity, the patch clamp technique has enabled scientists to perform manipulations with voltage dependent channels. These techniques include adjusting the polarity of the electrode in the patch pipette and altering the saline composition to moderate the free ion levels in the bath solution.
The patch clamp technique represents a major development in biology and medicine, since this technique allows measurement of ion flow through single ion channel proteins, and also allows the study of the single ion channel responses to drugs. In brief, in standard, patch clamp technique, a thin (app. 0.5-2 xcexcm in diameter) glass pipette is used. The tip of this patch pipette is pressed against the surface of the cell membrane. The pipette tip seals tightly to the cell and isolates a few ion channel proteins in a tiny patch of membrane. The activity of these channels can be measured electrically (single channel recording) or, alternatively, the patch clamp can be ruptured allowing measurements of the channel activity of the entire cell membrane (whole cell recording).
During both single channel recording and whole-cell recording, the activity of individual channel subtypes can be characterized by imposing a xe2x80x9cvoltage clampxe2x80x9d across the membrane. Through the use of a feedback loop, the xe2x80x9cvoltage clampxe2x80x9d imposes a voltage gradient across the membrane, and thereby voltage-sensitive channels can be activated.
The time resolution and voltage control in such experiments are impressive, often in the msec or even xcexcsec range. However, a major obstacle of the patch clamp technique as a general method in pharmacological screening has been the limited number of compounds that could be tested per day (typically no more than 1 or 2). Also, the very slow rate of solution change that can be accomplished around cells and patches may constitute a major obstacle.
A major limitation determining the throughput of the patch clamp technique is the nature of the feeding system, which leads the dissolved compound to perfused cells and patches. In usual patch clamp setups, cells are placed in large experimental chambers (0.2-2 ml), which are continuously perfused with a physiological salt solution. Compounds are then applied by changing the inlet to a valve connected to a small number of feeding bottles. However, a number of problems exist in the technique of the prior art. First, the number of different compounds is limited by the number of bottles that may be connected to the application system at one time. This number is usually less than 6. Second, the required volumes of the supporting liquid and the sample to be tested are critically high Third, the time needed to change the solute composition around cells and patches is long for usual patch clamp experiments. Fourth, the introduction and application of compounds to be tested usually involves a significant degree of manual manipulation and interruption, thus jeopardizing the integrity of the cell/pipette connection.
The development of sophisticated systems for local application of compounds to activate neurotransmitter regulated channels, like the U-tube and other systems, reduces the effective application times to 10-100 msec, which is often acceptable. However, the feeding systems which fill the U-tube are more inflexible and have lower capacity than those used for standard patch clamp. This presently limits the use of these procedures in the medical industry.
In WO 96/13721 an apparatus is disclosed for determining the effect of test samples of compounds on ion-transfer channels of a membrane, comprising an autosampler having a plurality of containers adapted to contain test samples in solution, and having means for automatically withdrawing said test samples from each of said containers and discharging them into a receptacle, a container adapted to contain a supporting liquid, a perfusion chamber adapted to receive a test sample in solution, a supporting liquid, and said membrane, said perfusion chamber comprising a reference electrode adapted to contact electrically a solution contained in said chamber, means for transporting said test samples in solution from the receptacle of said autosampler and said supporting liquid from its container into said perfusion chamber, and means for removing said test sample and said supporting liquid from said perfusion chamber, a patch pipette having an electrode therein, movably positioned over said perfusion chamber and adapted to provide a high electrical resistance seal with the surface of a patch of said membrane positioned within said chamber, and means electrically connected to said patch pipette electrode and said reference electrode for measuring the current passing through said electrodes before, during and after the introduction of said test sample into said perfusion chamber.
It is a disadvantage of the known apparatus that various manual operations are required to utilize the apparatus. For example, a new perfusion chamber with cells has to be positioned manually in the apparatus, a new pipette has to be mounted manually in the apparatus, a cell to be patch clamped is selected manually, the pipette tip is positioned manually on the membrane of the selected cell, the patch clamping is performed manually, etc.
It is an object of the present invention to provide an apparatus and a method for automatically connecting an electrode to a cell.
According to the invention, an electrode is moved towards one or more cells while an electrical parameter, such as voltage, current, resistance, conductance, inductance, capacitance, etc, is measured in an electrical circuit comprising the electrode and a chamber holding the cells. When the electrode enters nutrition liquid in the chamber holding the cells the parameter changes abruptly and also when the electrode enters a cell the parameter changes whereby contact between the electrode and the cell is detected. Upon such detection, movement of the electrode may be automatically stopped so that the electrode remains in contact with the cell membrane or, the movement may be continued a certain distance to insert the electrode in the internal part of the cell. For example, when the cell is a macroscopic cell, such as an oocyte, etc, measurements on the cell is typically performed with two electrodes inserted into the internal part of the cell.
It is a further object of the present invention to provide an apparatus and a method for automatically connecting an electrode to a cell membrane or an internal part of a cell, e.g. for recording electrical events in cell membranes of cells, that are positioned in a chamber, the apparatus and method including automation of at least one of the following steps:
positioning of a chamber with cells in an operating position,
positioning of an electrode or a pipette in an operating position adjacent the chamber,
selecting a cell to be connected to the electrode or pipette, e.g. to be patch clamped,
moving the electrode or the pipette to the selected cell, e.g moving the pipette tip to the cell membrane of the cell to be patch clamped,
patch clamping the cell, and
changing solutions in the chamber and recording electrical events of the selected cell, e.g. the cell patch.
The objects of the invention are fulfilled according to the invention by an automatic electrode positioning apparatus for connecting an electrode to a cell, comprising a chamber for holding cells, an electrode movably positioned adjacent the chamber, and positioning means for holding and positioning the electrode at desired positions in relation to the chamber. The apparatus may further comprise measurement means for determination of an electrical parameter and electrically connected to the electrode and the chamber and forming an electrical circuit comprising the electrode and the chamber. A controller controls the positioning means in response to the parameter determinations so that the electrode can be automatically connected to a selected cell.
In a group of cells, specific cells may have properties that makes it desirable to connect just such cells to the electrode. Thus, the apparatus may comprise selection means for selecting a specific cell to be connected to the electrode.
For example, the selection means may comprise the measurement means and the controller, the controller being further adapted to continue to control the positioning means so that the electrode scans the chamber with cells until a cell with a parameter within a predetermined range of the parameter is detected whereby that cell is selected.
The selection means may comprise imaging means for forming an image of cells in the chamber, digitizing means for recording and digitizing the image by dividing the image into pixels, the digitizing means being in operating communication with the imaging means, and a memory for storage of the digitized image and electrically connected to the digitizing means. For example, a grey tone image may be represented digitally by a digital image comprising pixels each of which has one pixel value representing the grey tone of the corresponding pixel. Similarly, a color image may be represented by a digital image comprising pixels each of which has three pixel values, one for each of the colors red, green, and blue.
The image may be displayed on a display unit, such as a CRT monitor, etc, constituting part of a user interface means of the selection means and an operator may then select a cell to be connected to the electrode, e.g. using a mouse, specific keyboard keys, etc.
The selection means may further comprise a processor that is connected to the memory and that is adapted for determination of the position of the selected cell from its position in the image. The controller may be electrically connected to the selection means and adapted to receive the position of the selected cell and to automatically control the positioning means in response to the received position in such a way that the electrode is positioned at the determined position of the selected cell.
Alternatively, the selection of a cell may be performed automatically. For this purpose, the processor may be further adapted for processing the digitized image for identification of cells in the chamber, selection of one cell to be connected to the electrode, and determination of the position of the selected cell in the chamber.
In a preferred embodiment of the invention for automatical patch clamping of a cell, the apparatus may comprise a pipette comprising the electrode and having a pipette tip adapted to provide a high electrical resistance seal with the surface of a cell membrane. Then, the positioning means are adapted for holding and positioning the pipette at desired positions in relation to the chamber, and the controller is adapted for automatically controlling the positioning means in response to the received position in such a way that the pipette tip is positioned at the determined position of the selected cell and provides a high electrical resistance seal with the cell membrane of the selected cell whereby an electrical parameter of the cell membrane can be determined without intervention of a human operator.
Thus, according to an important aspect of the invention, an automatic patch clamp apparatus for determination of an electrical parameter of a cell membrane is provided, comprising
the chamber for holding cells positioned in an operating position in the apparatus,
the pipette movably positioned adjacent the chamber in its operating position and having the pipette tip adapted to provide a high electrical resistance seal with the surface of a cell membrane,
the positioning means for holding and positioning the pipette at desired positions in relation to the chamber,
the selection means for selecting a cell, the cell membrane of which is to be connected with a high electrical resistance seal to the pipette tip, comprising
the processor adapted for
identification of cells in the chamber,
selection of one cell to be subsequently connected to the pipette tip, and
determination of the position of the selected cell in the chamber,
the controller that is electrically connected to the selection means and adapted to receive the determined position of the selected cell and to automatically control the positioning means in response to the received position in such a way that the pipette tip is positioned at the determined position of the selected cell and provides a high electrical resistance seal with the cell membrane of the selected cell, and
the measurement means for determination of an electrical parameter and electrically connected to the pipette tip and the chamber and forming an electrical circuit comprising the pipette tip and the chamber whereby an electrical parameter of the cell membrane can be determined without intervention of a human operator.
The chamber is typically constituted by a well in a chamber member. The cells may be grown on a coverslip, e.g. a 3 millimetre coverslip, which is positioned in the well. Preferably, the chamber is continuously perfused with an appropriate saline solution to prevent cells from drying and dying. Below, the chamber for holding cells is also denoted the perfusion chamber or the microperfusion chamber.
A reference electrode is preferably positioned in the chamber and in contact with liquid in the chamber to facilitate recording of electrical events in a cell membrane of a cell in the chamber.
The pipette is a patch pipette which typically is a thin (app. 0.5-2 xcexcm in diameter) glass pipette. During patch clamping of a cell membrane, the tip of the patch pipette is pressed against the surface of the cell membrane. The internal volume of the pipette may then be appropriately subjected to negative pressure for the pipette tip to seal tightly to the cell thereby forming a giga-seal between the pipette tip and the cell membrane. Preferably, the pipette has an electrode so that the activity of ion channels of the patched cell membrane can be measured electrically.
The negative pressure (suction) for the pipette tip may be generated by a modified Eppendorf oocyte injector. The injector may be modified to generate output pressures in the range from xe2x88x92300 hP to +300 hP in steps of 1 hP with a response time of approximately 10 msec. This fulfils patch clamping requirements.
The positioning means are adapted to receive and operationally engage with an electrode in a housing or a pipette and to move, preferably in three dimensions, the electrode or pipette to desired positions. The positioning means may comprise an electronic micromanipulator, such as a micromanipulator manufactured by Eppendorf.
The selection means may comprise any sensor suitable for detection of cells. For example, in a chamber with a very large number of cells, the electrode may be used to identify a cell by measurement of conductance between the electrode and the reference electrode of the chamber. When the electrode is lowered into the chamber, the conductance increases when it enters the liquid in the chamber and the conductance decreases again when the electrode connects to a cell membrane. A processor may be adapted for identifying cells by monitoring the conductance. In a sufficiently dense population of cells, only a small volume of the chamber need be searched by the electrode to identify a cell membrane by monitoring of the conductance and the first cell identified may be selected, e.g. for patch clamping. In this example, the position of the selected cell is known as the current position of the electrode.
The processor may comprise any computer such as a standard IBM compatible PC or a computer compatible with an Apple Macintosh (MAC).
In a chamber with a less dense population of cells, it is preferred to use an optical sensor for detection of cells and in a preferred embodiment also for selection of a cell, e.g. to be patched. Thus, the selection means may comprise imaging means, such as a microscope, etc, for forming an image of cells in the chamber and positioned adjacent the chamber, digitizing means, such as a hPCCD camera comprising a digital output, a video camera with a frame grabber, etc, for recording and digitizing the image by dividing the recorded image into pixels, each of which has a recorded intensity value, the digitizing means being in operating communication with the imaging means, a memory, such as the memory of a PC, for storage of the digitized image and electrically connected to the digitizing means.
The image may be displayed to an operator of the apparatus, e.g. on a CRT monitor, and the operator may select a cell utilizing a user interface means, such as a mouse, e.g. by moving a cursor with the mouse to a desired cell that may be selected by activation of a mouse button. The user interface means may comprise zoom means allowing selection of a specific pixel of the image of the selected cell whereby the corresponding point of the cell surface is selected to be the point of contact with the electrode or pipette.
Likewise, the operator may define the position of the tip of the electrode or pipette by moving the cursor to the corresponding position in the image. The pixel at the position of the cursor at activation of a mouse button may be selected as the position in the image of the tip of the electrode or pipette.
When the operator has selected a cell contact point to be connected with the electrode and/or to be patch clamped with the pipette, the apparatus may automatically move the electrode or pipette to the contact point, e.g. for performing automatical patch clamping.
In another embodiment of the invention, the processor is connected to the memory and further adapted for processing the digitized image for identification of cells in the chamber, selection of one cell to be subsequently connected to the pipette or electrode tip, and determination of the position of the selected cell in the chamber.
The processor may be adapted for processing the digitized image for identification of the pipette or electrode tip, and determination of the position of the pipette or electrode tip.
Any known and suitable image processing may be utilized to recognize and identify cells and/or the pipette or electrode tip, e.g. comprising edge detecting in order to identify the contour of cells and/or the pipette or electrode tip.
It is well known to represent an image digitally by dividing the image into a large number of segments, denoted pixels, and allocating digital intensity values, denoted pixel values, to each pixel. Typically, the image is divided into a matrix of rows and columns of pixels and the size of a digital image is then given by the number of pixels in a row and the number of pixels in a column. The pixel values are typically stored in an array of memory locations in a digital memory, each memory location corresponding to a pixel of the image. For example, a grey tone image may be represented digitally by a digital image comprising pixels each of which has one pixel value representing the grey tone of the pixel. Similarly, a colour image maybe represented by a digital image comprising pixels each of which has three pixel values, one for each of the calories red, green, and blue.
Further, it is well-known to process a digital image by forming a new digital image with the same number of pixels as the original image in which each of the new pixel values is generated by a linear or non-linear transformation of the corresponding original pixel value. For example, the new pixel value may be calculated from an algorithm, i.e. a spatial digital filter, of the original pixel value and pixel values of neighbouring pixel values, e.g. the new pixel value is the average of the original pixel values of the pixel in question and its eight neighbouring pixels.
It is presently preferred to identify cells utilizing spatial filtering comprising identifying original pixels and marking, e.g., a corresponding pixel of a new digital image with the same number of pixels as the original image, with a first mark when the original pixel is a pixel onto which a cell has been imaged, i.e. the pixel values of a specific number of neighbouring pixels to the original pixel in question, including the pixel in question. are lower than a selected threshold value.
The spatial filtering may further comprise identifying and marking with a second mark a group of neighbouring pixels marked with the first mark onto which a single cell is imaged by determining the number of pixels comprised in the group of neighbouring pixels and marking groups having a number of pixels within a predetermined range.
Still further, the spatial filtering may comprise identifying and marking with a third mark a group of neighbouring pixels marked with the second mark if the distance from pixels of the group of neighbouring pixels to pixels marked with the first mark is greater than a predetermined minimum distance.
The selected cell may be selected among cells that are imaged onto corresponding groups of neighbouring pixels marked with the third mark.
According to a preferred embodiment of the present invention, a set of geometrical parameters are calculated for cells with the third mark, such as maximum cell diameter, cell form factor, cell square extent, etc. The value of a selection parameter that is a function of the geometrical parameters is calculated for cells with the third mark and the selection of a cell is made based on calculated selection parameter values. For example the first cell having a calculated selection parameter value within a predetermined range may be selected or, the cell of the cells with the third mark with a selection parameter value closest to a desired value may be selected, etc. The selection parameter may be any arithmetic combination of the set of geometrical parameter, such as the product of maximum cell diameter, form factor and square extent.
The square extent of a cell is calculated by mathematically fitting a square around the cell and calculating the ratio of the number of pixels in the cell to the total number of pixels in the square. Thus, if dmax denotes the maximum width of a cell and A the area of the cell, the square extent is given by       A          d      max      2        .
If the circumference of the cell is denoted C, the form factor is given by             4      ⁢              xe2x80x83            ⁢      π      ⁢              xe2x80x83            ⁢      A              C      2        .
It is seen that the form factor has a value ranging from 0 to 1 and that the form factor for a circle is 1.
A predetermined selection parameter range may be determined by manually selecting appropriate cells, e.g. with a mouse and a cursor as described above, and calculating the selection parameter value of the selected cells. Upon selection of an appropriate number of cells, e.g. 25-40, the average and standard deviation of the cells may be calculated and during selection the cell with a parameter selection value closest to the average may be selected. Thus, different types of cells may lead to corresponding different selection parameter ranges.
The position of the centre pixel of the selected cell may constitute the determined position of the selected cell.
The signal-to-noise ratio of cell images may be enhanced considerably if the cells are made fluorescent or phosphorescent, e.g. by staining the cells with a fluorescent or phosphorescent dye, by implantation of a gene for a fluorescent or phosphorescent protein, e.g. the enhanced green fluorescent protein (EGFP). Further, the imaging means may comprise optical filters for transmission of radiation emitted from the cells and blocking other radiation. This may simplify the cell selection method described above. For example, when EGFP is used, cell selection may be reduced to the steps based on the desired intensity of the green colour marking cells with the first mark, calculating selection parameter values of cells with the first mark, and selecting the cell with the best selection parameter value.
It is presently preferred that the processor is adapted to identify pixels onto which the pipette tip or electrode is imaged utilizing spatial filtering of the image.
The spatial filtering may further comprise identifying and marking with a fourth mark a pixel as a pixel onto which the pipette or electrode tip is imaged when the pixel values of a specific number of neighbouring pixels to the pixel in question, including the pixel in question, are lower than a second threshold value.
Furthermore the spatial filtering may comprise identifying a line of pixels, each pixel on the line being positioned at the centre of pixels marked with the fourth mark and being positioned on the line of the pixel in question, and the position of the pipette tip may be determined as the position of an end pixel of the line of pixels.
In order to be able to test the influence of various compounds on a cell membrane for hours without intervention of an operator, it is necessary to be able to exchange the test chamber with the cell currently patch clamped with a new test chamber and a new patch pipette so that it is ensured that the test results always can be relied upon.
Thus, preferably, the apparatus further comprises a chamber member having a plurality of chambers for holding cells, and chamber member moving means for sequentially moving the chambers from a chamber storage position to the operating position.
It is preferred that a plurality of test cell cultures are introduced into the respective chambers of the plurality of chambers before performing tests of compounds. Preferably, the cells are grown on a layer of protein on a coverslip that is positioned in a respective chamber prior to testing of compounds.
The term xe2x80x9coperating positionxe2x80x9d means that the positioning means for holding and positioning the pipette and the chamber are positioned in relation to each other in such a way that a selected cell in the chamber can be patch clamped to the pipette tip. The chamber member with the chambers may be moved in relation to fixed,positioning means or the positioning means may be moved in relation to a fixed chamber member.
Preferably, the present apparatus comprises chamber member moving means for moving a chamber from a first storage position in which position the chamber may be supplied with a liquid, e.g. a saline solution, sustaining living cells, to the operating position, and to another storage position when a cell in another chamber is to be patched.
The chamber member moving means may comprise the positioning means as they may be adapted to push and/or pull the chamber member to desired positions.
The chamber member may comprise a chamber member memory means for storage of data and the apparatus may comprise means for reading the data contained in the chamber member memory means.
The data may comprise an expiry date of the chamber member, identification data, calibration data, etc.
In order to facilitate correct movement of a chamber from the unused storage position to the operational position, the chamber member moving means may be controlled in any known manner. This known manner may be a movement controlled by time, length of movement, angle of rotation of the motor shaft, or optical, electrical or mechanical means may be provided for determining when the desired chamber is in its operating position and for terminating the movement.
The chamber member may comprise chambers arranged in a rectangular array of columns and rows of chambers, the chamber member being moved in relation to the positioning means along axis of rows and columns when a new chamber is moved to its operating position, or, the chambers may be arranged in circular arrays, e.g. on a turn-table, the circular array being rotated in relation to the positioning means when a new chamber is moved to its operating position.
The chamber member may be mounted on a mounting plate which can be moved in an x-y plane by two electromotors and the chamber member may be rotatable, e.g. by a third motor, about an axis perpendicular to the x-y plane so that the individual chambers can be positioned in their respective operating positions by rotation of the chamber member. The imaging system may further comprise an inverted microscope forming an assembly with the mounting plate. The position of the focus plane of the microscope may be adjustable thus, facilitating autofocussing prior to initiation of pipette and cell recognition.
Holders for holding electrodes or pipettes may be positioned with the electrodes or pipettes in an array, e.g. in two rows, on a member that is positioned on the mounting plate.
Alternatively, the chamber member may further comprise holders for holding pipettes.
The positioning means may be further adapted to selectively withdraw a pipette from its holder and to insert the pipette into its holder, e.g. when the corresponding chamber is in its operating position.
The apparatus may further comprise means for supplying liquid, such as a saline solution, to the chambers of the chamber member.
Preferably, the means for supplying liquid to the chambers of the chamber member comprises means for supplying a first liquid to the chambers in a storage position and a second liquid to the chamber in the operating position.
The apparatus may further comprise suction means for removing excess liquid flowing through the chambers.
According to the present invention the patch clamp technique is combined with the use of an autosampler, a combination which was unobvious at the outset to a person skilled in the art, especially due to the fact that the technique is sensitive to disturbances, such as vibrations and electrical noise arising from the autosampler, so that it would at best have been considered an impossible or inoperative combination.
It is a further object of the present invention to provide and adapt apparatus for automatic drug handling and application, and to utilize the apparatus in an electrophysiological system for screening of chemical substances or compounds to measure their effect on ion channel transfer, the novel system providing high throughput and low fluid volume requirements.
It is yet a further object of the present invention to reduce the needed amount of any chemical compound for testing as well as to increase the rate of screening, thereby providing the first electrophysiology test system suitable for commercial pharmaceutical company screening.
It is still further an object to provide novel microperfusion chamber structures having microperfusion chambers of extremely low volume.
It is still an additional object of the invention to provide novel methods for carrying out patch clamp technique utilizing the apparatus of the present invention. The foregoing and other objects, advantages, and characterizing features of the invention will become apparent from the following description of certain illustrative embodiments thereof considered together with the accompanying drawings, wherein like reference numerals signify like elements throughout the various figures.
What we believe to be our invention, then, inter alia, comprises the following, singly or in combination:
An apparatus for determining the effect of test samples of compounds on ion-transfer channels of a membrane, comprising:
an autosampler having a plurality of containers adapted to contain test samples in solution, and having means for automatically withdrawing said test samples from each of said containers and discharging them into a receptacle,
a container adapted to contain a supporting liquid,
a perfusion chamber adapted to receive a test sample in solution, a supporting liquid, and said membrane, said perfusion chamber comprising a reference electrode adapted to contact electrically a solution contained in said chamber,
means for transporting said test samples in solution from the receptacle of said autosampler and said supporting liquid from its container into said perfusion chamber, and means for removing said test sample and said supporting liquid from said perfusion chamber,
a patch pipette having an electrode therein, movably positioned over said perfusion chamber and adapted to provide a high electrical resistance seal with the surface of a patch of said membrane positioned within said chamber, and
means electrically connected to said patch pipette electrode and said reference electrode for measuring the current passing through said electrodes before and after the introduction of said test sample into said perfusion chamber,
such apparatus wherein the means for transporting test samples and supporting liquid to said perfusion chamber includes a U-tube mounted over said perfusion chamber and having an aperture therein for releasing a test sample and its supporting liquid into said perfusion chamber; and
apparatus for determining the effect of test samples of compounds on ion-transfer channels of a membrane, comprising:
an autosampler having a plurality of containers adapted to contain test samples in solution, and having means for automatically withdrawing said test samples from each of said containers and discharging them into a receptacle,
a container adapted to contain a supporting liquid,
a microperfusion chamber adapted to receive a test sample in solution, a supporting liquid, and said membrane, the volume of said microperfusion chamber being about 5 microliters to about 50 microliters, said microperfusion chamber comprising a reference electrode adapted to contact electrically a solution contained in said chamber,
means for transporting a test sample in solution from the receptacle of said autosampler and said supporting liquid from its container into said microperfusion chamber and means for removing said test sample and said supporting liquid from said microperfusion chamber,
a patch pipette having an electrode therein. movably positioned over said microperfusion chamber and adapted to provide a high electrical resistance seal with the surface of a patch of said membrane positioned within said chamber, and
means electrically connected to said patch pipette electrode and said reference electrode for measuring the current passing through said electrodes before and after the introduction of a test sample into said microperfusion chamber;
such apparatus wherein the volume of said microperfusion chamber is in the range of about 10 microliters to about 15 microliters; such
apparatus wherein the volume of said microperfusion chamber is in the range of about 10 microliters to about 12 microliters; such
apparatus having means for aspirating waste liquid from said microperfusion chamber; such
apparatus wherein said means of said autosampler for automatically withdrawing said test samples from said containers and discharging them into a receptacle comprises a syringe pump and a needle connected thereto; and such
apparatus wherein a tubular coil is positioned in series with said means for transporting said test sample for quantitatively determining the volume of the sample introduced into said microperfusion chamber.
Also, a microperfusion chamber assembly comprising: a base, an aperture in said base, and transparent means over the bottom of said base. said aperture and said transparent means cooperating to define the side walls and the bottom of a microperfusion chamber, a reference electrode arranged to contact a liquid in said chamber, means for introducing a liquid into said chamber, means for aspirating liquid from said chamber, and means for electrically connecting said reference electrode to an electrical measuring device;
such a microperfusion chamber assembly wherein said transparent means forming the bottom of said chamber is a transparent coverslip; such
a microperfusion chamber assembly wherein said base comprises silver having a coating of a silver halide deposited over at least the surface of the aperture defining the side walls of said microperfusion chamber, thereby providing a reference electrode in said base adapted to make electrical contact with a liquid contained in said chamber; such
a microperfusion chamber assembly wherein said base comprises silver having a coating of silver chloride deposited over at least the surface of the aperture defining the side walls of said microperfusion chamber; such
a microperfusion chamber assembly wherein said base comprises silver having a coating of silver chloride deposited over its entire surface including the side walls of said chamber, such
a microperfusion chamber assembly wherein said microperfusion chamber is cylindrical, frustoconical, or ellipsoid; such
a microperfusion chamber assembly wherein the volume of said microperfusion chamber is about 5 microliters to about 50 microliters; such
a microperfusion chamber assembly wherein the volume of said microperfusion chamber is about 10 microliters to about 15 microliters; such
a microperfusion chamber assembly wherein the volume of said microperfusion chamber is about 10 microliters to about 12 microliters; and
a microperfusion chamber assembly comprising a base formed of a non-electrically conductive material, an aperture provided in said base, and transparent cover means at the bottom of said base, said aperture and said transparent cover means cooperating to define the side walls and bottom of a microperfusion chamber, a reference electrode mounted in said base extending through a side wall of said microperfusion chamber and arranged electrically to contact a liquid contained in said chamber, means for introducing a liquid into said chamber, means for aspirating liquid from said chamber, and means for electrically connecting said reference electrode to an electrical measuring device;
such a microperfusion chamber assembly wherein said base comprises a plastic material; such
a microperfusion chamber assembly wherein said plastic material is polymethyl methacrylate, polystyrene, polyvinyl chloride, or polycarbonate; such
a microperfusion chamber assembly wherein said electrode comprises a silver wire surrounded by a mixture of particulate silver and a silver halide; such
a microperfusion chamber assembly wherein said electrode comprises a silver wire surrounded by a mixture of particulate silver and silver chloride; such a microperfusion chamber assembly wherein said particulate silver and silver chloride are affixed to said silver wire; such
a microperfusion chamber assembly wherein said means for introducing a liquid into said microperfusion chamber is a groove provided in said base cooperating with said transparent cover means to define a channel, the proximal end of said channel communicating with said microperfusion chamber, and an aperture provided at the top of said base communicating with the distal end of said channel and adapted to have an inflow duct connected thereto for introducing a liquid into said microperfusion chamber;
such a microperfusion chamber assembly wherein the volume of said microperfusion chamber is about 5 micro-liters to about 50 microliters; such
a microperfusion chamber assembly wherein the volume of said microperfusion chamber is about 10 microliters to about 15 microliters; and such
a microperfusion chamber assembly wherein the volume of said microperfusion chamber is about 10 microliters to about 12 microliters.
Also, a method for determining the effect of test samples of compounds on ion-transfer channels of a membrane contained in a perfusion chamber, wherein a patch pipette having an electrode therein has its tip engaged in a gigaohm seal with the surface of said membrane, and wherein a reference electrode is provided in said chamber adapted to contact a solution contained in said chamber, comprising
continuously or periodically introducing a supporting liquid containing ions, the transfer characteristics of which are to be determined as a baseline reference, into said chamber,
periodically loading one of said test samples dissolved in the same supporting liquid into said chamber utilizing an autosampler having a plurality of containers holding test samples in solution, and
measuring the electrical current flowing in an electrical measuring means circuit connected between said pipette electrode and said reference electrode both before and after introduction of said test sample into said perfusion chamber, and repeating the procedure; such
a method wherein said supporting liquid is continuously introduced into said perfusion chamber and the excess aspirated, and wherein said autosampler is programmed periodically to introduce a test sample into the flowing stream of said supporting liquid to flow therewith into said perfusion chamber; such
a method wherein said perfusion chamber is a microperfusion chamber having a volume of 5 microliters to about 50 microliters; such
a method wherein the volume of said microperfusion chamber is about 10 microliters to about 15 microliters; such
a method wherein the volume of said microperfusion chamber is about 10 microliters to about 12 microliters; such
a method wherein said supporting liquid is periodically introduced into said perfusion chamber and the excess aspirated, and wherein said autosampler is programmed periodically to cause a test sample to flow into said perfusion chamber; such
a method wherein some of said containers in said autosampler contain said supporting liquid and wherein said autosampler is programmed periodically to introduce said supporting liquid and said test sample sequentially into said perfusion chamber; such
a method wherein only small volumes of both test samples and supporting liquid are employed, wherein said perfusion chamber is a microperfusion chamber having a volume of about 5 to about 50 microliters, wherein some of the containers in said autosampler contain said supporting liquid, and wherein said autosampler first aspirates a volume of supporting liquid from one of said containers and causes the supporting liquid to enter said microperfusion chamber, and wherein said autosampler subsequently aspirates a test sample from one of said containers and causes the test sample to enter said microperfusion chamber and to replace said supporting liquid in said chamber, and wherein an electrical measurement is made both when said supporting liquid and when said test sample are present in said microperfusion chamber; such
a method wherein the volume of said microperfusion chamber is about 10 microliters to about 15 microliters; such
a method wherein the volume of said microperfusion chamber is about 10 microliters to about 12 microliters; such
a method wherein an external electrical current is imposed on said electrodes to bring the reference current to the desired value; and such
a method wherein an external electrical current is imposed on said electrodes to bring the reference current to the desired value.